Specific Vash Compound (SVC) Inhibitors For Cardiac Disease

Abstract

The present invention relates to a compound of formula (I): or a pharmaceutically acceptable salt and / or solvate thereof for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction and / or for improving cardiac function in a subject in need thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to new specific Vash compounds (SVC) for use in the prevention and / or treatment of heart diseases, in particular heart failure and cardiomyopathy. BACKGROUND

[0002] Heart failure (HF) is defined as the inability of the heart to provide adequate blood flow to the body's organs and tissues. Heart failure is often characterized by diastolic dysfunction, or an insufficient filling of the ventricles due to the inability of the myocardium to fully relax. Diastolic dysfunction is a hallmark of hypertrophic cardiomyopathy (HCM) (Maron et al., 1995) and heart failure with preserved ejection fraction (HFpEF). These diseases are characterized by prolonged and impaired diastolic phase of the left ventricle (LV), slow LV filling, and increased diastolic LV stiffness.

[0003] The prevalence of heart failure continues to rise, and its significant mortality highlights the need for new therapies. In particular, heart failure with preserved ejection fraction (HFpEF) currently has limited treatment options, even though it is estimated to account for about half of all heart failure cases. Many HFpEF cases manifest as slow myocardial relaxation, leading to abnormal pump function. The slow diastolic relaxation of the heart in HFpEF is also associated with increased internal resistance due to microtubule (MT) densification and stabilization post-translational modifications such as a-tubulin de-tyrosination (Chen et al., 2018). Therefore, targeting MTs to decrease overall cardiomyocyte stiffness can increase the rate of relaxation without increasing energy consumption.

[0004] Microtubules (MTs) are major cytoskeletal components present in all eukaryotic cells. They polymerize from a- and b-tubulin heterodimers and act as mechanical sensors, translating varying contractile forces into intracellular signals. In addition, MTs act as anti-compression elements, providing mechanical impedance to cardiac myocyte contraction. They act as viscoelastic resistance elements that can impair sarcomere shortening, thus impairing cardiac function, particularly in disease states associated with MT proliferation. Post-translational modifications (PTMs) of MTs alter their mechanical properties and binding interactions (Roll-Mecak, 2020). De-tyrosination is a PTM of a-tubulin that has recently been shown to influence MT-dependent mechanical transduction in cardiac and skeletal muscle diseases (Kerr J.P et al., 2015). This specific PTM is significantly increased in patient tissues and preclinical models of heart disease (Chen et al., 2018). In failing myocardium, the maladaptation of cardiomyocytes to chronic stress is associated with densification of the MT network and increased VASH protein abundance and / or de-tyrosination activity. Densification of the MT network and disease-associated de-tyrosination activity exacerbate, mechanically interfere with the function of cardiomyocytes by impairing contraction, leading to compensatory mechanisms and significant remodeling of the myocardium.

[0005] There remains an urgent need to develop safe and effective treatment options for heart failure, particularly those that minimize the increased risk of ischemia or arrhythmia associated with current expensive palliative treatments. Over the past five years, researchers have identified modified cardiac myocyte (CM) microtubules (MTs) as a novel therapeutic target for treating HF. There is a significant microtubular densification and increased de-tyrosination activity in different sources of human and murine HF (Tsutsui H. et al., 1993; Cheng G. et al., 2008), which leads to cardiac myocyte stiffening and impaired contractile performance. Reversing this post-translational modification by forced expression of a counteracting enzyme named tubulin tyrosine ligase (TTL) is sufficient to reduce the stiffness and improve contractility of human and murine failing CMs and myocardium. However, the use of viral expression vectors in cardiovascular applications remains complex. Notably, for cardiovascular medicine knowledge, modulating the de-tyrosination status by expressing TTL can improve cardiac mechanical properties independent of calcium cycling, which greatly reduces the risk of this novel therapy to “de-tyrosinate”.

[0006] Studies have shown that de-tyrosination-reducing gene therapy can improve cardiac function in heart failure. Although cardiac gene therapy has great potential, it faces many obstacles, especially in large animals, achieving safe and effective cardiac transduction is a major challenge.

[0007] At the same time, inhibition of de-tyrosination enzymes shows considerable therapeutic potential. The vasohibin enzymes (VASH1 and VASH2) work with or without their partner small vasohibin binding protein (SVBP) to remove C-terminal tyrosine residues from polymerized MT a-tubulin (Van der Laan S. et al., 2019). VASH1 was recently identified as the main de-tyrosination enzyme in human and murine myocardium, and it was found that VASH1 KD was sufficient to reduce the stiffness of failing human CMs, increase the fractional shortening, and accelerate their contraction and relaxation. Notably, VASH1 KD resulted in significantly faster relaxation of CMs in patients with heart failure with preserved ejection fraction (HFpEF), independent of transient changes in Ca 2+ Because VASH1 does not bind and sequester free tubulin, VASH1 KD does not exhibit significant MT depolymerization in CMs (Chen, C.Y. et al., 2020), but rather specifically reduces dTyr while maintaining network dynamics and density. Preventing VASH2-dependent de-tyrosination significantly limited the reduction in left ventricular ejection fraction after acute myocardial infarction in mice without affecting infarct size or cardiac remodeling (Yu X. et al., 2021).

[0008] Currently available VASH inhibitors have drawbacks, but are improving. Parthenolide (PTL) is a highly reactive sesquiterpene lactone, is an FDA-approved chemotherapeutic drug, does indeed directly inhibit VASH activity (Aillaud, C. et al., 2017), although only at high concentrations. In CM, 10 mM PTL (lower than its IC 50 ) treatment for 2 hours moderately reduces dTyr (30-40%) (Kerr, J.P. et al., 2015; Robison P. et al., 2016; Schuldt, M. and Kuster, 2020), but has many potential off-target effects limiting its long-term use, including modulating NF-kB, STAT3 and JNK signaling pathways (Caporizzo, M.A., Chen, C.Y. and Prosser, B.L., 2019), and cannot be translated into product development.

[0009] Therefore, there is still a need to provide VASH compound (SVC) inhibitors that offer more specificity.

[0010] In 2017, an epoxide-Y (Epo-Y) was described and used in targeted chemical proteomics screening to identify a de-tyrosinase complex and to act as a VASH inhibitor more than 10 times stronger than PTL (Aillaud, C. et al., 2017).

[0011] But the inventors have recently developed SVC inhibitors that improve VASH inhibition by 10 to 1000 times compared to Epo-Y and that easily cross the cell membrane to effectively reduce dTyr in cells and improve cardiomyocyte function. The inventors tested these compounds on ZSF1 obese rats, which are considered the best rodent model of heart failure with preserved ejection fraction (HFpEF) as they present 3 major comorbidities associated with human HFpEF, namely hypertension, obesity and diabetes. And they demonstrated that these compounds effectively improved diastolic function. In particular, they demonstrated that SVC inhibitors significantly reduced the stiffness of ZSF1 obese cardiomyocytes and improved their contraction and relaxation kinetics relative to ZSF1 lean controls. And advantageously, when administered in vivo by intravenous injection or orally, these compounds are well tolerated and effective in reducing de-tyrosination. SUMMARY

[0012] Therefore, the present invention relates to a method for treating a patient suffering from heart failure and / or cardiomyopathy, in particular a patient suffering from heart failure with reduced or preserved ejection fraction (e.g. HFrEF or HFpEF, respectively), and / or for improving diastolic function in a patient suffering from heart failure and / or cardiomyopathy, in particular a patient suffering from heart failure with reduced or preserved ejection fraction (e.g. HFrEF or HFpEF, respectively), comprising administering to said patient a therapeutically effective amount of a VASH compound (SVC) inhibitor. 2+Compositions and methods to reduce cardiac stiffness and improve contractility in an independent manner. The compositions and methods provided herein can be combined with other therapies ("combination therapy"), including those involving Ca 2+ independent of those involving Ca

[0013] A first aspect of the present invention relates to a method for improving cardiac function in a subject in need thereof, comprising administering to a subject in need thereof a compound of formula (I)

[0014]

[0015] or a pharmaceutically acceptable salt and / or solvate thereof,

[0016] wherein

[0017] X is or -NH-CH2-

[0018] R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, which is unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl,

[0019] R is O-R 2 wherein R 2 is C1-C6 aliphatic chain, which is optionally substituted, or C1-C6 alkyl-aryl,

[0020] R 3 is OH or O-C1-C6 aliphatic chain,

[0021] Y is -(CH2) m wherein m = 2 or

[0022] R 5 is C(O)OH or O-C1-C6 alkyl, and

[0023] R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain.

[0024] In other words, the present invention also relates to a compound of formula (I):

[0025]

[0026] or pharmaceutically acceptable salts and / or solvates thereof,

[0027] wherein

[0028] X is or -NH-CH2-

[0029] R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, which is unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl,

[0030] R is O-R 2 wherein R 2 is C1-C6 aliphatic chain, which is optionally substituted, or C1-C6 alkyl-aryl,

[0031] R 3 is OH or O-C1-C6 aliphatic chain,

[0032] Y is -(CH2) m wherein m = 2 or

[0033] R 5 is C(O)OH or O-C1-C6 alkyl, and

[0034] R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain,

[0035] for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction and / or for use in improving cardiac function in a subject in need thereof.

[0036] Another aspect of the present application is a method for treating a cardiovascular disease selected from the group consisting of heart failure, cardiomyopathy and myocardial infarction-induced cardiac dysfunction in a subject in need thereof, said method is administering to a subject in need thereof an effective dose of a compound of formula (I)

[0037]

[0038] or pharmaceutically acceptable salts and / or solvates thereof,

[0039] wherein

[0040] X is or -NH-CH2-

[0041] R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, which is unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl,

[0042] R is O-R 2 wherein R 2 is C1-C6 aliphatic chain, which is optionally substituted, or C1-C6 alkyl-aryl,

[0043] R 3 is OH or O-C1-C6 aliphatic chain,

[0044] Y is -(CH2) m wherein m = 2 or

[0045] R 5 is C(O)OH or O-C1-C6 alkyl, and

[0046] R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain.

[0047] Another aspect of the present application is a combination therapy, wherein the compound (I) described in the present application is combined with the current standard treatment regimen for heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0048] Another aspect of the present application is the use of the above-mentioned compound (I) or of conjugates comprising fragments of the compound of formula (I) (which fragments can or can not be linked to a biological molecule, and which biological molecule can optionally be labeled) as research tools for research and development activities in cardiac function diseases or heart failure.

[0049] DETAILED DESCRIPTION

[0050] DEFINITIONS

[0051] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0052] The terms "a" or "an," as used herein, mean one or more. Accordingly, the singular form "a" or "an" used herein includes plural references unless context clearly dictates otherwise. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0053] The words "comprise," "comprises" and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist," "consist of," and "consisting of are to be interpreted exclusively rather than inclusively. While various embodiments in the specification are presented using "comprising" language, where other embodiments are presented using "consisting of" or "consisting essentially of" language, the related embodiments are also intended to be interpreted using the "consisting of" or "consisting essentially of" language.

[0054] The term "about" includes a variance range of ±10% unless otherwise noted.

[0055] As used herein, the term "therapy" refers to any regimen, method, and / or drug that can be used to prevent, manage, treat, and / or ameliorate a disease, disorder, or condition in a patient. The patient can be at risk of contracting the disease, disorder, or condition, or be suspected of having contracted the disease, disorder, or condition. Alternatively, the patient can have been diagnosed with the disease, disorder, or condition. Non-limiting examples of therapies include administration of a composition (e.g., a pharmaceutical composition), physical therapy, psychotherapy, etc. Therapies also include the possibility of combining several therapies. In this case, the various therapies can be sequential, simultaneous, or mixed. When the therapy comprises administration or administration of a composition, the composition is administered in an amount, manner, and / or mode effective to treat or prevent the disease, disorder, or condition in the patient. The therapy can require multiple administrations of the composition.

[0056] As used herein, the term "treating" or "treatment" means the improvement of a disease, disorder, or condition of a patient, which can be observed at the clinical, histological, and / or biochemical level. The term "treating" or "treatment" specifically includes ameliorating a clinical, histological, and / or biochemical symptom or parameter associated with a disease, disorder, or condition of a patient, or inhibiting, reducing, or delaying progression or worsening of a disease, disorder, or condition of a patient, including secondary injuries caused by the disease, disorder, or condition, to a statistically significant or a degree detectable by one skilled in the art. In some embodiments, a treatment regimen is assessed on a population basis, such that a therapy is considered to "treat" a particular disease, disorder, or condition if a statistically significant improvement in the disease, disorder, or condition of patients is observed in a population of people with the disease, disorder, or condition.

[0057] As used herein, the terms "prevent," "prevention," and "preventing" refer to reducing the risk of acquiring or developing a given disease, disorder, or condition in a subject. The terms "prevent," "prevention," and "preventing" also include delaying the onset and / or reducing the frequency and / or intensity of a clinical, histological, and / or biochemical symptom or parameter associated with the given disease, disorder, or condition. In some embodiments, a prevention is assessed on a population basis, such that a therapy is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the risk of acquiring or developing the particular disease, disorder, or condition, and / or a statistically significant delay in the onset and / or a statistically significant reduction in the frequency and / or intensity of a clinical, histological, and / or biochemical symptom or parameter associated with the disease, disorder, or condition is observed in a population of people susceptible to the disease, disorder, or condition.

[0058] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration, or implanting a slow-release device, such as a microosmotic pump, into a subject. Administration can be by any route, including parenteral and transmucosal administration (e.g., buccal, sublingual, palatine, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous drip, transdermal patches, and the like.

[0059] “Co-administer” means that the composition described herein is administered at the same time, immediately prior to, or immediately after administration of one or more other therapies. In certain embodiments, the composition is specifically targeted (e.g., via direct injection) to the heart.

[0060] The term “combination” as used herein refers to any possible arrangement of the various components (e.g., a compound of Formula (I) according to the application and another therapeutic regimen). Such arrangements include mixtures of the components and separate combinations for administration either simultaneously or sequentially. The application includes combinations comprising equimolar concentrations of the components as well as combinations where the concentrations vary greatly. It will be appreciated by those skilled in the art that the optimal concentration of each component in the combination can be determined.

[0061] “Current standard treatment regimen for heart failure, cardiomyopathy and cardiac dysfunction induced by myocardial infarction” refers to an accepted treatment regimen for a cardiovascular disease selected from heart failure, cardiomyopathy and cardiac dysfunction induced by myocardial infarction, such as the regimen listed in the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure (McDonagh T.A et al., 2021).

[0062] “a subject in need thereof’ refers to an animal, preferably a mammal including a human. In a particular and preferred embodiment, the subject in need thereof is a human suffering from or susceptible to a cardiac disease, in particular heart failure, cardiomyopathy or cardiac dysfunction induced by myocardial infarction.

[0063] “Heart failure” is a condition that occurs when the heart cannot pump enough blood to meet the body’s needs. This happens if the heart does not fill with enough blood. It also happens when the heart is too weak to pump blood normally.

[0064] Heart failure can develop suddenly (acute) or over time as the heart weakens (chronic). It can affect one side or both sides of the heart. Left- and right-sided heart failure can have different causes. Often, heart failure is caused by another disease that damages the heart, including coronary heart disease and subsequent myocardial infarction, inflammation of the heart, high blood pressure, cardiomyopathy, or arrhythmia. Heart failure can damage the liver or kidneys. Other diseases it can cause include pulmonary hypertension or other heart diseases such as arrhythmia, heart valve disease, and cardiac arrest.

[0065] “Cardiomyopathy” refers to problems with the heart muscle that make it harder for the heart to pump blood. There are many types and causes of cardiomyopathy, and it can affect people of all ages. Depending on the type of cardiomyopathy, the heart muscle can become thicker, stiffer, or larger than normal. This weakens the heart and can lead to irregular heartbeats, heart failure, and even sudden cardiac death. It includes dilated, hypertrophic, ischemic, genetic, and idiopathic cardiomyopathy.

[0066] In one particular embodiment, the heart failure or cardiomyopathy in the present application comprises chronic heart failure or cardiomyopathy.

[0067] In another particular embodiment, the heart failure or cardiomyopathy in the present application comprises acute heart failure (AHF) syndrome, which includes post-myocardial infarction, post-cardiectomy status, post-cardiac arrest, hypertensive crisis, acute manifestations of non-ischemic cardiomyopathy, and acute exacerbations of chronic cardiomyopathy of various etiologies.

[0068] The term “stereoisomer” as used in the present application refers to configurational stereoisomers, more particularly to optical isomers.

[0069] In the present application, optical isomers are in particular produced by different positions in space of the substituents attached to X. Thus, the carbon atom or the nitrogen atom of the X group to which the substituents are attached represents a chiral or asymmetric center. Accordingly, optical isomers which are not mirror images of each other are named “diastereomers”, and optical isomers which are non- superimposable mirror images are named “enantiomers”.

[0070] An equimolar mixture of the two enantiomers of a chiral compound is named as a racemic mixture or racemate.

[0071] In the context of the present application, depending on the position of the substituents attached to the X group, the compounds of the present application can be diastereomers of the following configurations (S, S), (R, R), (S, R) or (R, S):

[0072]

[0073] When the position of the substituents is not specified in the compounds, the compounds correspond to any one of the above diastereomers or a mixture of the diastereomers.

[0074] In one particular embodiment, the compounds for use in the present application are diastereomers of the configuration (S, S).

[0075] Within the framework of the present application, the term “pharmaceutical composition” refers to a composition having preventive and therapeutic properties.

[0076] For the purposes of the present application, the term "pharmaceutically acceptable" means useful in preparing a pharmaceutical composition and generally safe and non-toxic in the amounts employed in pharmaceutical uses.

[0077] Within the framework of the present application, the term "pharmaceutically acceptable salts and / or solvates" means salts and / or solvates of the compounds as described hereinbefore which are pharmaceutically acceptable, and which possess the pharmacological activity of the corresponding compound.

[0078] Pharmaceutically acceptable salts include:

[0079] (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, and the like; or formed with organic acids such as acetic acid, benzenesulfonic acid, fumaric acid, glucoheptonic acid, glycolic acid, glutamic acid, glyceric acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, succinic acid, dibenzoyl-L-25-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid and trifluoroacetic acid, and the like, and

[0080] (2) base addition salts formed when an acidic proton present in the compound is replaced by a metal ion, for example, an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or coordinates with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

[0081] The solvates of the compounds of the present application acceptable in therapeutic use include conventional solvates such as those formed from the presence of a solvent in the final step of the preparation of the compounds of the present application. For example, mention can be made of solvates due to the presence of water (these solvates are also called hydrates) or ethanol.

[0082] The term "C x -C y The term "aliphatic chain" denotes a straight chain or branched hydrocarbon chain which is completely saturated or contains one or more unsaturated but is not aromatic, comprising x to y carbon atoms, in particular 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. According to the present application, the term "aliphatic chain" includes substituted or unsubstituted, straight chain or branched alkyl, alkenyl or alkynyl groups.

[0083] The term "C1-C6 alkyl" as used in the present application means a straight chain or branched monovalent saturated hydrocarbon chain containing from 1 to 6 carbon atoms, including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl (also known as tert-butyl), n-pentyl, n-hexyl and the like.

[0084] The term "aryl" as used in the present invention refers to aromatic hydrocarbon radicals preferably comprising 6 to 12 carbon atoms and comprising one or more condensed rings, such as, but not limited to, phenyl or naphthyl. Advantageously, it is a phenyl group.

[0085] The term "Ci-C6-alkylaryl" as used in the present invention refers to alkyl radicals as described above which are substituted by an aryl radical as described above, respectively. Advantageously, "Ci-C6-alkylaryl" is benzyl.

[0086] In the context of the present invention, "optionally substituted" means that the group is optionally substituted by one or more substituents, in particular selected from the group consisting of Ci-C6-alkyl, NR a R b , COR c , CO2R d , CONR e R f , ORg, N + R h R i R j , wherein R a to R j are independently of each other H, Ci-C6-alkyl or aryl, preferably H or Ci-C6-alkyl.

[0087] The term "peptide coupling" refers to a chemical reaction between an amine function and a carboxylic acid function. The peptide coupling will advantageously be performed in the presence of a coupling agent such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI), 2-(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1 H-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-(7-azabenzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), (benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) or propylphosphonic anhydride; optionally in combination with an additive or a base such as N-hydroxy-succinimide (NHS), N-hydroxy-benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazol (HOOBt), 1 -hydroxy-7-azabenzotriazole (HAt), N-hydroxysulfosuccinimide (sulfo-NHS), dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA) or N-methylmorpholine (NMM).

[0088] The term "VASH or Vasohibin (enzyme)" used in the present invention refers to a tubulin carboxypeptidase (TCPase) involved in the microtubule de-tyrosination mechanism associated with muscular dystrophy, in particular heart failure, cardiomyopathy and cardiac dysfunction induced by myocardial infarction.

[0089] The term "biomolecule" refers to a molecule having biological properties. In the context of the present invention, it refers to a protein, a polypeptide, a biomarker, such as but not limited to a photo-labeling agent (e.g. rhodamine, coumarin derivative, cyanine derivative or fluorescein), an affinity probe (e.g. biotin), or an E3 ubiquitin ligase recruiting agent (e.g. thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096 or d9A-2).

[0090] The term "prodrug" relates to a usually pharmacologically inactive or less active derivative of an active drug which undergoes a biotransformation in a cell or in vivo, by chemical or enzymatic cleavage, to release the active drug. In the context of the present invention, "pharmacologically inactive or less active derivative" means that the prodrug does not have the relevant activity to inhibit the active site of VASH in an in vitro environment. However, they are active in cellular and in vivo assays because such tests allow to provide the transformation required for the active drug. Prodrugs can offer a number of advantages over the parent drug, such as increased cellular penetration, solubility, enhanced stability, improved bioavailability, reduced side effects and better selectivity. The activation of a prodrug can involve a number of enzymes, including but not limited to oxidoreductases such as CYP450 and DT-diaphorase, and hydrolytic enzymes such as carboxylesterases and beta-glucuronidases.

[0091] In the context of the present invention, the prodrug compounds described in the present disclosure can be inactive in vitro. However, the prodrug compounds have increased cellular penetration. Upon cellular penetration, the prodrug compounds are hydrolyzed to provide the corresponding active drug, i.e. the potent VASH inhibitor.

[0092] Therefore, a first aspect of the present invention is a method for improving cardiac function in a subject in need thereof, comprising administering to the subject in need thereof a compound of formula (I)

[0093]

[0094] or a pharmaceutically acceptable salt and / or solvate thereof,

[0095] wherein

[0096] X is or -NH-CH2-

[0097] R 1 is or NR1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, which is unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl,

[0098] R is O-R 2 wherein R 2 is C1-C6 aliphatic chain, which is optionally substituted, or C1-C6 alkyl-aryl,

[0099] R 3 is OH or O-C1-C6 aliphatic chain,

[0100] Y is -(CH2) m -, wherein m = 2 or

[0101] R 5 is C(O)OH or O-C1-C6 alkyl, and

[0102] R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain.

[0103] In other words, the present application relates to a compound of formula (I):

[0104]

[0105] or a pharmaceutically acceptable salt and / or solvate thereof,

[0106] wherein

[0107] X is or -NH-CH2-

[0108] R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, which is unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl,

[0109] R is O-R 2 wherein R 2is C1-C6 aliphatic chain, which is optionally substituted, or C1-C6 alkyl-aryl,

[0110] R 3 is OH or O-C1-C6 aliphatic chain,

[0111] Y is -(CH2) m -, wherein m = 2 or

[0112] R 5 is C(O)OH or O-C1-C6 alkyl, and

[0113] R 6 is NH-CH(R 7 )-(CH2) n -R 8 , wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain, for use in the prevention and / or treatment of heart failure, cardiomyopathy, myocardial infarction-induced cardiac dysfunction, and / or for use in improving cardiac function in a subject in need thereof.

[0114] Compounds of formula (I) used in the application

[0115] The compounds according to the present application can be in the form of a stereoisomer or a mixture of stereoisomers, e.g. a mixture of enantiomers or diastereomers, in particular a racemic mixture. According to a specific embodiment, the compound of formula (I) is in the form of one diastereomer of the configuration (S,S), of the configuration (R,R), of the configuration (S,R) or of the configuration (R,S) as described above.

[0116] Formula (I) as described herein encompasses active compounds (i.e. drugs) and prodrugs thereof.

[0117] In particular, the term "prodrug" according to the present application refers to a compound of formula (I) wherein R 3 is not OH.

[0118] Preferably, the prodrug according to the present application is a compound of formula (I) wherein R 3 is O-C1-C6 aliphatic chain, e.g. O-C1-C6 alkyl, in particular O-ethyl, O-isopropyl or O-tert-butyl.

[0119] In the context of the present application, the term "drug" refers to a compound of formula (I) wherein R 3 is OH.

[0120] Typically, R 3together with the adjacent carboxyl group form an ester group. When converted from the prodrug to the drug, the ester group present in the prodrug is converted to the corresponding carboxylic acid in the drug. 3 The ester formed with the adjacent carboxyl group is typically hydrolyzed to the corresponding carboxylic acid.

[0121] In the context of the present application, the drug differs from its corresponding prodrug in that the ester group present in the prodrug is converted to the corresponding carboxylic acid in the drug. Preferably, the prodrug comprises only one ester group 3 together with the adjacent carboxyl group form an ester group. Thus, the drug corresponding to a given prodrug typically has the same molecular formula as the prodrug, except for the R 3 group. In other words, in a given prodrug and its drug, the substituents R 1 , X and R are typically identical, respectively. Alternatively, the prodrug can comprise two or more ester groups. In this case, all ester groups are converted to carboxylic acids in the corresponding drug.

[0122] Preferably, the compound of formula (I) is in the form of the diastereoisomer of configuration (S, S) and corresponds to the following formula (I-A):

[0123]

[0124] wherein X, R, R 1 and R 3 are as described in the present disclosure.

[0125] In a more preferred embodiment, the compound of formula (I-A) corresponds to the following enantiomer (I-A’):

[0126]

[0127] wherein X, R, R 1 and R 3 are as described in the present disclosure.

[0128] In a preferred embodiment, X is preferably

[0129] In another particular embodiment, X is -NH-CH2-.

[0130] According to the present application, R is OR 2 .

[0131] According to some embodiments, R 2 is a C1-C6 aliphatic chain, such as a C1-C6 alkyl or C1-C6 alkyl-aryl group, said aliphatic chain or alkyl-aryl group being optionally substituted. When R 2 is a C1-C6 alkyl, such as a methyl or ethyl group, it is in particular unsubstituted (unsubstituted), or substituted with a phenyl group. More preferably, R 2is C1-C6 alkyl, for example ethyl, or C1-C6 alkyl-aryl, for example benzyl.

[0132] According to some other embodiments, R 3 is OH or O-C1-C6 aliphatic chain. More preferably, R 3 is OH or O-C1-C6 alkyl, in particular O-ethyl, O-isopropyl or O-tert-butyl.

[0133] According to some embodiments, R 1 is NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl, said alkyl being optionally substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl. In such embodiments, R 1 may in particular be NH-C1-C6 alkyl, said alkyl being, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl or tert-butyl, substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl, in particular by C(O)-methyl, C(O)-ethyl, C(O)-isopropyl, C(O)-tert-butyl, or C(O)-NH-methyl, C(O)-NH-ethyl, C(O)-NH-isopropyl, or C(O)-NH-tert-butyl.

[0134] According to a preferred embodiment, R 1 is Preferably

[0135] The compound of formula (I) can be a compound of formula:

[0136] wherein R, R 3 , R 5 , R 6 , X and Y are as described in the present disclosure.

[0137] Preferably, the compound of formula (I) is a compound of formula (I-A a ) having the following configuration:

[0138] wherein R, R 3 , R 5 , R 6 , X and Y are as described in the present disclosure.

[0139] More preferably, the compound of formula (I) is a compound of formula (I-A’ a ) having the following configuration:

[0140] wherein R, R 3 , R 5 , R 6 , X and Y are as described in the present disclosure.

[0141] Y can in particular be In such an embodiment, R 5 is preferably O-Ci-C6alkyl, such as O-methyl or O-ethyl, in particular O-ethyl.

[0142] Alternatively, Y can be -(CH2) m -, wherein m is an integer from 0 to 6, preferably m is 1 or 2, more preferably m is 2. In embodiments wherein Y is -(CH2) m , R 5 is preferably C(O)OH.

[0143] According to some embodiments, R 6 is O-Ci-C6aliphatic chain, such as O-methyl or O-ethyl, in particular O-ethyl, in particular when Y is .

[0144] According to other specific embodiments, R 6 is NH-CH(R 7 )-(CH2) n -R 8 , in particular when Y is -(CH2) m -, in particular -(CH2)2-. In such embodiments, R 7 is preferably H, R 8 is preferably aryl, such as phenyl, n is 1, 2 or 3, preferably 1.

[0145] According to a preferred embodiment, when R 3 is OH, R 1 is is preferably

[0146] In a preferred embodiment, the compound of formula (I) is a compound of formula (I-A’a) as described above, wherein X, R and R 3 are as described above, R 1 is is preferably wherein:

[0147] -Y is -(CH2) m -, m is as described above and in particular m is 2, R 5 is C(O)OH, R 6 is NH-CH(R 7 )-(CH2) n-R 8 , R 7 is H, R 8 is aryl, for example phenyl, and n is 1, 2 or 3, or

[0148] -Y is R 5 is O-Ci-C6alkyl, for example O-methyl or O-ethyl, and R 6 is O-Ci-C6aliphatic chain, for example O-methyl or O-ethyl.

[0149] In another more preferred embodiment, the compound of formula (I) has the formula (I-A’), wherein X is R 1 is NR 1a R 1b , wherein R 1a is preferably H and R 1b is preferably Ci-C6alkyl, for example methyl, ethyl, n-propyl, n-butyl or tert-butyl, preferably ethyl, isopropyl or tert-butyl, R is OR 2 , wherein R 2 is preferably Ci-C6alkyl, in particular ethyl or Ci-C6alkyl, in particular benzyl, and R 3 is OH or O-Ci-C6alkyl, in particular O-ethyl, O-isopropyl or O-tert-butyl.

[0150] In another more preferred embodiment, the compound of formula (I) has the formula (I-A’), wherein X is R 1 is NR 1a R 1b , wherein R 1a is preferably H and R 1b is preferably Ci-C6alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl or tert-butyl (tert-butyl), preferably ethyl, isopropyl or tert-butyl, R is OR 2 , wherein R 2 is preferably Ci-C6aliphatic chain substituted by phenyl, and R 3 is O-Ci-C6alkyl, in particular O-ethyl, O-isopropyl or O-tert-butyl.

[0151] In a particular embodiment, when X is then

[0152] R 1 is preferably or NR1a R 1b wherein R 1a is H and R 1b is unsubstituted or substituted with C(O)-O-C1-C6alkyl or C(O)-NH-C1-C6alkyl,

[0153] R is O-R 2 wherein R 2 is C1-C6aliphatic chain, which is unsubstituted or substituted with phenyl, or C1-C6alkyl-aryl,

[0154] R 3 is OH or O-C1-C6aliphatic chain,

[0155] Y is -(CH2) m - wherein m = 2 or

[0156] R 5 is C(O)OH or O-C1-C6alkyl, and

[0157] R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6aliphatic chain.

[0158] In some preferred embodiments,

[0159] R 1 is and

[0160] R is O-R 2 wherein R 2 is C1-C6aliphatic chain, which is unsubstituted or substituted with phenyl,

[0161] R 3 is OH or O-C1-C6aliphatic chain,

[0162] Y is -(CH2) m - wherein m = 2 or

[0163] R 5 is C(O)OH or O-C1-C6alkyl, and

[0164] R 6 is NH-CH(R 7 )-(CH2) n-R 8 wherein R 7 is H and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6aliphatic chain.

[0165] In another specific embodiment, when X = -NH-CH2-

[0166] R 1 is preferably NR 1a R 1b wherein R 1a is H and R 1b is C1-C6alkyl which is unsubstituted or substituted by C(O)-NH-C1-C6alkyl,

[0167] R is O-R 2 wherein R 2 is C1-C6alkyl-aryl, and

[0168] R 3 is O-C1-C6aliphatic chain.

[0169] In a specific embodiment, the compound of formula (I) according to the application is a prodrug as described above, in particular selected from the following compounds:

[0170]

[0171] and the pharmaceutically acceptable salts and / or solvates thereof.

[0172] In another specific embodiment, the compound of formula (I) according to the application is a drug as described above, in particular the following compounds:

[0173]

[0174] or a pharmaceutically acceptable salt and / or solvate thereof.

[0175] In a specific and preferred embodiment, the compound of formula (I) according to the application is selected from the following compounds

[0176]

[0177] the pharmaceutically acceptable salts and / or solvates thereof, and

[0178] conjugates thereof.

[0179] Methods of preparing compounds of formula (I)

[0180] The compound of formula (I) as described above, or a pharmaceutically acceptable salt and / or solvate thereof, can be obtained by a process comprising the following steps:

[0181] (a) reacting a compound of formula (II) with a compound of formula (III):

[0182]

[0183] wherein R Z is R 1 or OH,

[0184] (b) optionally, converting R Z which is OH into R 1 as described above.

[0185] The compound of formula (III) can be obtained according to methods well known to the person skilled in the art.

[0186] The reaction between the compounds of formula (II) and (III) is in particular a peptide coupling as described above.

[0187] The compound of formula (II) is in particular

[0188] Optionally, additional protection / deprotection and / or functionalization steps well known to the person skilled in the art can occur before or after the reaction between the compounds of formula (II) and (III) to obtain a compound of formula (I) as described above with suitable substituents.

[0189] In particular, when R Z is OH, the compound obtained from step (a) is subjected to a peptide coupling with to obtain a compound of formula (I) wherein R 1 is

[0190] One or more peptide couplings performed in the process for the preparation of a compound of formula (I) are in particular achieved in the presence of PyAOP as coupling agent. Preferably, also the base DIEA is used.

[0191] The peptide coupling can be performed on solid support, in particular by using resins linked to the reagent (one of the amine moiety or the acid moiety). These methods are known to the person skilled in the art.

[0192] Conjugates comprising a fragment of a compound of formula (I) linked to a biomolecule

[0193] The present application also relates to the use of a conjugate comprising a fragment of a compound of formula (I) as described above, which fragment is linked to a biological molecule, such as a polypeptide, a protein, a biomarker (e.g. a light labelling agent, such as rhodamine, a cyanine derivative or fluorescein), an affinity probe (such as biotin), or an E3 ubiquitin ligase recruiter (including but not limited to thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096 or d9A-2).

[0194] The term "fragment of a compound of formula (I)" means that one of the terminal of a compound of formula (I) is modified by binding to a biological molecule (e.g. through a linker). Typically, R 1 The group is thus modified to allow said binding. For example, in a conjugate according to the application, the fragment of a compound of formula (I) means the following moiety:

[0195]

[0196] wherein, is a single bond between the fragment and the rest of the conjugate, and R, R 3 , X, Y and R 5 as described in the present disclosure.

[0197] According to one particular embodiment, the conjugate according to the application is of formula (I') below:

[0198]

[0199] or a pharmaceutically acceptable salt and / or solvate thereof,

[0200] wherein,

[0201] B is a biological molecule, such as a polypeptide, a protein, a biomarker (e.g. a light labelling agent), or an E3 ligase recruiter (such as thalidomide),

[0202] L is a linker, and

[0203] R, R 3 , X, Y and R 5 as described above.

[0204] According to one preferred embodiment, the conjugate of formula (I') is a conjugate of formula (I'-A) having the following configuration:

[0205]

[0206] In particular, the conjugate of formula (I') is a conjugate of formula (I'-A a ) having the following configuration:

[0207]

[0208] In the conjugate of formula (I’), in particular of formula (I’-A a ) X is preferably

[0209] R is preferably OR 2 , R 2 is advantageously C1-C6 alkyl, in particular ethyl, or C1-C6 alkyl-aryl, in particular benzyl.

[0210] R 3 is preferably OH or O-C1-C6 alkyl, in particular O-ethyl, O-isopropyl or O-tert-butyl.

[0211] Y can be -(CH2) m - or

[0212] When Y is -(CH2) m -, in particular -(CH2)2-, R 5 is preferably C(O)OH, R 6 is preferably NH-CH2-(CH2) n -R 8 , wherein R 8 is advantageously aryl, for example phenyl, and n is advantageously 1, 2 or 3, in particular 1.

[0213] When Y is , R 5 is preferably O-C1-C6 alkyl, for example O-ethyl.

[0214] According to some embodiments, the linker L corresponds to a divalent radical derived from a C1-C 12 aliphatic chain, in which one or more methylene units are replaced by a structural linker selected from arylene or the fragments -O-, -S-, -C(=O)-, -SO2- or -N(C1-C6 alkyl)-, wherein said aliphatic chain is unsubstituted or substituted by one or more groups selected from halogen, OH, C1-C6 alkyl and / or C1-C6 alkyl aryl, such as benzyl.

[0215] In one embodiment, the biomolecule is an affinity probe, and the conjugate of formula (I’) can be:

[0216]

[0217] wherein the compound of formula (I) is linked to biotin.

[0218] In another embodiment, the biomolecule is a photo-labeling agent, and the conjugate of formula (I’) can be:

[0219]

[0220] wherein the compound of formula (I) is linked to rhodamine.

[0221] Pharmaceutical compositions used in the application

[0222] The present application also relates to a pharmaceutical composition for the prevention or treatment of a cardiac disease, comprising at least one pharmaceutically acceptable excipient and at least one compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as described above.

[0223] The present application also relates to a pharmaceutical composition for the prevention or treatment of a cardiac disease, comprising at least one conjugate as described above, such as a conjugate of formula (I’), or a pharmaceutically acceptable salt and / or solvate thereof, and at least one pharmaceutically acceptable excipient.

[0224] In some embodiments, the pharmaceutical composition for use according to the present application is intended for administration by oral or parenteral (including, but not limited to, intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial), preferably oral or intravenous administration. Other modes of delivery include, but are not limited to, the use of liposomal preparations, intravenous drip, transdermal patches, etc. The active ingredients can be mixed with conventional pharmaceutical carriers to be used in unit form for administration to animals, preferably mammals, including humans, in the form of tablets, capsules, etc.

[0225] These compositions can be delivered transdermally via a topical route, and can be formulated as applicator sticks, solutions, suspensions, lotions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral formulations include tablets, pills, powders, capsules, liquid formulations, lozenges, cachets, gels, slurries, suspensions, and the like, suitable for ingestion by the patient. Solid form formulations include powders, tablets, capsules, cachets, suppositories, and dispersible granules. Liquid form formulations include solutions, suspensions, and emulsions, such as water or water / propylene glycol solvents. The compositions can also contain other ingredients to achieve sustained release and / or comfort. These components include high molecular weight anionic mucin-mimicking polymers, gelling polysaccharides, and finely dispersed pharmaceutical carrier matrices. Compositions can also be delivered as microspheres for slow release in vivo. For example, microspheres can be administered by intradermal injection of drug-containing microspheres that slowly release subcutaneously; as biodegradable and injectable gel formulations; or as microspheres for oral administration. In another embodiment, the compositions can be delivered by using liposomes that fuse with or are endocytosed by cell membranes, i.e., by attaching receptor ligands to the surface of the liposomes, which bind to cell membrane surface membrane protein receptors, triggering endocytosis. By using liposomes, especially when the liposome surface carries receptor ligands specific for target cells, or is otherwise preferentially directed to a particular organ (e.g., the heart), the compositions can be delivered precisely to target cells in vivo.

[0226] Combination administration refers to simultaneous or sequential administration of the compounds, alone or in combination with other active substances (such as those disclosed below), one or more compounds or drugs.

[0227] Combination therapy or combination products

[0228] The compounds of formula (I) can be combined with other current standard treatment regimens for heart failure, cardiomyopathy or cardiac dysfunction induced by myocardial infarction, such as those listed in the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure (McDonagh T.A. et al., 2021).

[0229] The most appropriate combination will be determined by the subject and the heart disease by the heart failure specialist. Combinations with SVC compounds (as dual pharmacophores) can be intuitively attractive:

[0230] 1) Administration with a positive inotropic agent with a different and complementary mechanism of action (MoA). This can be a beta agonist (such as dobutamine, dopamine or adrenaline), a phosphodiesterase type 3 inhibitor (such as milrinone) or an alternative positive inotropic drug (such as levosimendan) to enhance the overall positive inotropic rescue strategy in cardiogenic shock states without increasing the energy demand.

[0231] 2) with a negative inotropic agent which should be beneficial but is poorly tolerated due to its negative inotropic effect. In patients with severe cardiomyopathy or cardiogenic shock, this can be a combination with a beta-adrenergic blocker, but also with a myosin inhibitor such as mavacamten or other emerging drugs.

[0232] The present application therefore also relates to a method of treating a cardiovascular disease selected from heart failure, cardiomyopathy and cardiac dysfunction induced by myocardial infarction in a subject in need thereof, which comprises administering to the subject in need thereof an effective dose of a compound of formula (I) as disclosed above, in combination with a current standard treatment regimen for heart failure, cardiomyopathy or cardiac dysfunction induced by myocardial infarction. In particular, the current standard treatment regimen for heart failure is selected from a positive inotropic agent or a negative inotropic agent, in particular (i) a positive inotropic agent selected from a beta agonist, a phosphodiesterase type 3 inhibitor and an alternative positive inotropic agent, or (ii) a negative inotropic agent selected from a beta-adrenergic blocker and a myosin inhibitor.

[0233] Another object of the present application is a combination product comprising:

[0234] a) a compound of formula (I) as described in the present application or a pharmaceutical composition as described in the present application, and

[0235] b) a current standard treatment regimen for heart failure, cardiomyopathy or cardiac dysfunction induced by myocardial infarction.

[0236] In particular, the current standard treatment regimen for heart failure is selected from a positive inotropic agent or a negative inotropic agent, in particular (i) a positive inotropic agent selected from a beta agonist, a phosphodiesterase type 3 inhibitor and an alternative positive inotropic agent, or (ii) a negative inotropic agent selected from a beta-adrenergic blocker and a myosin inhibitor.

[0237] Use of compounds and pharmaceutical compositions in cardiac disease

[0238] The compound of formula (I), the pharmaceutically acceptable salts and / or solvates thereof and / or the pharmaceutical composition according to the present application act as VASH inhibitors, meaning that they are able to inhibit the peptidase activity of VASH catalyzing the de-tyrosination of microtubules. When this VASH peptidase activity is dysregulated, in particular abnormally increased, it induces a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0239] X in the compound of formula (I) is when it is a significant irreversible VASH inhibitor, covalently binding to the VASH enzyme.

[0240] The present application relates to the use of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof for the preparation of a medicament for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0241] In other words, the present application relates to the use of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof for the preparation of a medicament intended for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0242] In other words, the present application relates to the use of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof for the preparation of a medicament intended for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0243] In particular, the present application relates to the use of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof for the prevention and / or treatment of heart failure, cardiomyopathy, cardiac dysfunction induced by myocardial infarction and / or for the improvement of cardiac function in a subject in need thereof.

[0244] In other words, the present application relates to a method for the improvement of cardiac function in a subject in need thereof, comprising the administration to a subject in need thereof of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof.

[0245] In particular, the compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof improves the kinetics of failing cardiomyocytes, including increasing relaxation velocity, increasing contractility, and / or decreasing viscoelasticity.

[0246] In other words, the present application relates to a method for the treatment of a cardiovascular disease selected from heart failure, cardiomyopathy and cardiac dysfunction induced by myocardial infarction in a subject in need thereof, comprising the administration to a subject in need thereof of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof.

[0247] In other words, the present application relates to a method for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction, comprising the administration to a subject in need thereof of an effective dose of a compound according to the present application of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof.

[0248] According to another aspect, the present application relates to a pharmaceutical composition according to the present application for use as a medicament for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0249] In other words, the present application relates to the use of a pharmaceutical composition according to the present application for the preparation of a medicament for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0250] In other words, the present application relates to the use of a pharmaceutical composition according to the present application for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction.

[0251] In other words, the present application relates to a method for the prevention and / or treatment of a cardiac disease, in particular heart failure, cardiomyopathy and / or cardiac dysfunction induced by myocardial infarction, comprising administering to a subject in need thereof an effective dose of a pharmaceutical composition according to the present application.

[0252] In certain embodiments, the subject in need is suffering from heart failure associated with failing cardiomyocytes.

[0253] In certain embodiments, the subject in need is suffering from heart failure with reduced ejection fraction (HFrEF).

[0254] In certain embodiments, the subject in need is suffering from heart failure with preserved ejection fraction (HFpEF).

[0255] In certain embodiments, the pharmaceutical composition of the present application is used to decrease viscoelasticity and / or to accelerate cardiomyocyte relaxation.

[0256] In some embodiments, the pharmaceutical composition of the present application is used to increase the speed of cardiomyocyte relaxation by Ca 2+ independent mechanisms.

[0257] In certain embodiments, the pharmaceutical composition of the present application is used to decrease cardiomyocyte stiffness, to improve myocyte relaxation, to increase ventricular compliance and / or to decrease diastolic pressure.

[0258] In certain embodiments, the pharmaceutical composition of the present application is used to treat a patient suffering from diastolic dysfunction.

[0259] In certain embodiments, the pharmaceutical composition of the present application is used to treat a chronic cardiac disease, in particular chronic heart failure or cardiomyopathy.

[0260] In certain embodiments, the pharmaceutical composition of the present application is used to treat an acute heart failure (AHF) syndrome, which includes post-myocardial infarction, post-cardiotomy status, post-cardiac arrest, hypertensive crisis, acute manifestations of non-ischemic cardiomyopathy and acute exacerbations of chronic cardiomyopathy of various etiologies.

[0261] In certain embodiments, the pharmaceutical composition of the application is used for the treatment of a patient suffering from systolic dysfunction.

[0262] In certain embodiments, the pharmaceutical composition of the application is used for the preparation of a medicament for the treatment of failing cardiomyocytes.

[0263] In certain embodiments, the pharmaceutical composition of the application is used for the preparation of a medicament for improving the dynamics of failing ventricular cardiomyocytes.

[0264] In certain embodiments, the pharmaceutical composition of the application is used for the preparation of a medicament for reducing the viscoelasticity of cardiomyocytes.

[0265] Research tool use

[0266] Another aspect of the application is the use of a compound (I) or of a conjugate comprising a fragment of a compound of formula (I), which fragment is linked or not to a biological molecule, and which biological molecule is optionally labeled, as a research tool for research and development activities in cardiac dysfunction or heart failure.

[0267] In some embodiments, the application relates to the use of a compound of formula (I) according to the application or of a conjugate thereof, as a research tool in research and development activities selected from:

[0268] - in vitro methods for studying the role of de-tyrosinated tubulin in cardiac diseases, such as its onset, aggressiveness and progression,

[0269] - and related kits for performing said screening assays and methods.

[0270] These R&D activities are examples of illustrative but non-limiting uses of said compound of formula (I) or of a conjugate thereof.

[0271] Said compound of formula (I) can be conjugated with, for example but not limited to, a fluorescent dye, a UV photosensitive dye, HRP, alkaline phosphatase, biotin. BRIEF DESCRIPTION OF DRAWINGS

[0272] Figure 1 : High magnification image of immunohistochemical staining of de-tyrosinated tubulin in healthy myocardial tissue. High magnification shows individual cardiomyocytes. The purple staining is specific de-tyrosinated tubulin.

[0273] Figure 2 : Structure of a specific Vash compound (SVC) which is a potent inhibitor.

[0274] Figure 3: In vitro de-tyrosination assay using recombinant human VASH1 (hVASH1) and SVBP complex. ELISA-based de-tyrosination enzyme activity assay method. Dose response accurately measures the inhibitory effect of a compound on the target. Of note, SVC_02 is a prodrug. When tested using the recombinant enzyme, the compound is inactive in its prodrug form. It contains a specific group that effectively masks the second group necessary to bind the enzyme.

[0275] Figure 4 : Immunofluorescence staining of wild-type human cells and VASH double knock-out. The background signal generated by SVC_01 treatment is comparable to the signal observed in VASH 2KO cells, indicating that the target is fully occupied. The level of acetylated tubulin is not affected by SVC_01 treatment, as highly similar staining pattern and levels are observed under different conditions.

[0276] Figure 5 : Immunofluorescence method to detect the level of tubulin de-tyrosination in human cells. A dose-dependent decrease in tubulin de-tyrosination in human cells is observed after 2 hours of treatment with all SVCs. The IC 50 is 10 nM.

[0277] Figure 6 : In vitro de-tyrosination assay using recombinant human enzyme in the absence or presence of SVC_01 or BzI SA (specific carboxypeptidase A inhibitor). Carboxypeptidase A (CPA) is able to hydrolyze the peptide bond of C-terminal residues with aromatic or aliphatic side chains. No cross-reaction between different enzymes is observed. The reaction is stopped using SDS-page laemli buffer and Western blotting is performed using specific antibodies.

[0278] Figure 7 : Assay using active recombinant human rhinovirus (HRV) type 14 3C cysteine protease in the absence (negative) or presence (increasing doses) of SVC_01. HRV is a P1’ site-preferential non-aromatic residue protease. The optimal recognition site for human rhinovirus (HRV) type 14 3C protease is LEVLFQ / GP, with a glycine at the P1’ site. SVC_01 does not decrease the activity of this cysteine protease.

[0279] Figure 8: Assay using active Cathepsin B cysteine protease. The graph shows the activity of Cathepsin B in the presence of the commercial Cathepsin B & L inhibitor FF-FMK (positive control) or in the presence of increasing doses of SVC_01. Cathepsin B is a cysteine protease that prefers aromatic residues at the P1' site. Like VASH, Cathepsin B prefers aromatic residues at the P1' site. This is considered the best control for us to validate specificity.

[0280] Figure 9 : Cell viability assay comparing the effect of microtubule-binding drugs (parthenolide, paclitaxel) versus SVC_01 on human cells. The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of the yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. In contrast to microtubule-binding drugs that show severe toxicity, SVC_01 does not affect cell proliferation.

[0281] Figure 10 : Mitochondrial activity assay using the Agilent Seahorse CellMito Stress Test kit in the absence or presence of SVC_01 [20 mM]. This assay is a widely accepted and recognized standard assay for assessing mitochondrial energy function. Multiple parameters are accessible in one assay, including basal respiration, ATP-linked respiration, maximal and spare respiratory capacity, and non-mitochondrial respiration. This assay provides insights into mechanisms of mitochondrial dysfunction and allows users to investigate functional differences between cell types, candidate drugs, and genetic or biochemical interventions. High exposure of SVC_01 does not change mitochondrial activity.

[0282] Figure 11 : Potassium currents mediated by the hERG channel (referred to as hERG currents) were recorded using the whole-cell configuration patch-clamp technique. The percentage of inhibition values represents the mean of the percentage values calculated from the individual differences in the amplitude of the hERG tail current of each cell with the extracellular solution (steady-state phase). The mean percentage of inhibition at the highest test concentration (i.e., 30 pmol / L) is reported in the table. For each compound, a 6-point concentration-response curve was established.

[0283] Figure 12: Dose-dependent decrease in de-tyrosination following inhibition of VASH in primary cardiomyocytes isolated from Sprague Dawley rats. A) Representative Western blot and B) Quantification of de-tyrosination and a-tubulin levels following incubation of freshly isolated adult rat cardiomyocytes with the indicated concentrations of compound SVC_01 for 2 hours at 37°C. N = 4 Sprague Dawley rats.

[0284] Figure 13 : Following inhibition of VASH, primary cardiomyocytes isolated from healthy Sprague Dawley rats exhibit a dose-dependent modest improvement in contractile and relaxation kinetics during unloaded shortening. A) Resting sarcomere length, B) Fraction shortening, C) Contraction time, and D) Relaxation time in healthy isolated rat cardiomyocytes. n = 50-60 cardiomyocytes per group from N = 3 Sprague Dawley rats. One-way ANOVA.

[0285] Figure 14 : Western blots of myocardial tissue from Wistar Kyoto (WKY), ZSF1 lean, and ZSF1 obese rats were prepared. Protein samples were prepared and separated by PAGE and immunoblot analysis was performed with the indicated specific antibodies.

[0286] Figure 15 : Immunofluorescence images of de-tyrosinated and tyrosinated tubulin in ZSF-1 obese cardiomyocytes following incubation with / without SVC_01 for 2 hours at 37°C, showing almost complete disappearance of de-tyrosinated tubulin and preservation of the tyrosinated tubulin network.

[0287] Figure 16 : Inhibition of VASH or overexpression of TTL improves contractile and relaxation kinetics in primary HFpEF cardiomyocytes isolated from WKY, ZSF1 lean, or ZSF1 obese rats during unloaded shortening. A) Contraction amplitude, B) Contraction time, and C) Relaxation time in isolated rat cardiomyocytes. n ~ 90 cardiomyocytes per group from N = 3 ZSF1 lean or obese rats; n = 60 cardiomyocytes from N = 2 WKY rats. Two-way ANOVA with factors genotype and treatment.

[0288] Figure 17: Inhibition of VASH or overexpression of TTL reduces the transverse stiffness of primary cardiomyocytes isolated from ZSFl lean or ZSFl obese rats. A) Plot of stiffness (elastic modulus) as a function of nanoindentation velocity in ZSFl-lean and B) ZSFl-obese cardiomyocytes. C) Quantification of Emin (elastic stiffness), Emax (stiffness at maximum indentation velocity) and D) Delta E (viscoelasticity index) in ZSFl-lean and obese cardiomyocytes. ZSFl obese cardiomyocytes are more viscoelastic than their lean counterparts and this viscoelastic stiffness can be reduced by SVC_01 and overexpression of TTL.

[0289] Figure 18 : Inhibition of VASH reduces the longitudinal stiffness of primary cardiomyocytes isolated from ZSFl lean or ZSFl obese rats. A) Diastolic stiffness at 10%, 200 ms strain when applying a strain along the long axis of isolated cardiomyocytes (elastic modulus at initial peak strain); B) Steady-state stiffness after strain and hold and C) Stress relaxation (index of viscoelasticity) of ZSFl lean and obese cardiomyocytes. ZSFl obese cardiomyocytes are more viscoelastic than their lean counterparts and this viscoelastic longitudinal stiffness can be reduced by SVC_01 treatment.

[0290] Figure 19 : A) Western blot from myocardial tissue shows that de-tyrosinated tubulin is effectively reduced after a single injection of SVC_02 to 8-week-old Sprague Dawley rats. B) Quantification of de-tyrosination levels (D1 / a-tubulin ratio) in vehicle (n=4) and VASHi-treated rats (n=3).

[0291] Figure 20 : Western blot from myocardial tissue shows that de-tyrosinated tubulin is effectively reduced after two intravenous injections of SVC_02 to 30-week-old ZSFl-lean and obese rats.

[0292] Figure 21 : VASHi acutely improves diastolic function in HFpEF rats. A) Echocardiographic assessment of left ventricular diastolic function in ZSFl lean and obese animals + / - SVC_02 treatment. In ZSFl obese rats, the improvement of E / A ratio (marker of left ventricular function) and mitral deceleration time after inhibition of VASH indicate an improved diastolic function with faster ventricular relaxation. B) Hemodynamic measurements of left ventricular diastolic function in ZSFl lean and obese animals + / - SVC_02 treatment. Improved tau (left ventricular diastolic time constant) and dP / dTmin (rate of ventricular relaxation) indicate a faster ventricular relaxation after inhibition of VASH in ZSFl obese rats.

[0293] Figure 22: Volcano plot of differentially expressed extracellular and tubulin-related transcripts in ZSF1 obese vs. lean rats using NanoString analysis.

[0294] Figure 23 : Immunofluorescence imaging for detection of detyrosinated tubulin on LV tissue and assessment of changes in detyrosinated tubulin abundance following SVC-02 treatment.

[0295] Figure 24 : Quantification of immunofluorescence imaging analysis of LV tissue to assess changes in microtubule density (SVC-02 as VASHi).

[0296] Figure 25 : Quantification of Western blot analysis from myocardial tissue showing effective reduction of detyrosinated tubulin following two i.v. injections of SVC_02 to 30-week-old WKY, ZSF1-lean and obese rats.

[0297] Figure 26 : Patient demographics of donors.

[0298] Figure 27 : Average sarcomere shortening of cardiomyocytes isolated from non-failing (NF) and failing (HF) human hearts treated with vehicle (black) and SVC-02 (VASHi) (grey).

[0299] Figure 28 : Inhibition of VASH (SVC-02) improves relaxation of isolated failing human cardiomyocytes.

[0300] Figure 29 : Hematoxylin and eosin staining showing normal appearance of different organs in Swiss mice following systemic treatment with SVC-02 compared to control vehicle-treated animals.

[0301] Figure 30 : Immunofluorescence detection of detyrosination levels of tubulin in human cells. A dose-dependent reduction of detyrosination of tubulin in human cells was observed following 2 hours of treatment with all SVCs. The IC 50 of SVC_06 in cells was 2.1 nM.

[0302] Figure 31 : Quantification of Western blot analysis of isolated myocardial tissue following oral administration of SVC-06 to Sprague-Dawley rats.

[0303] Figure 32 : In vitro detyrosination using recombinant human VASH1 and VASH2 enzymes was determined at increasing concentrations of SVC_01. SVC-01 effectively inhibited both VASH1 and VASH2.

[0304] Figure 33 Adult human cardiomyocytes were treated with vehicle or SVC-01 (inhibitor). Protein lysates were analyzed by western blot to assess levels of tubulin de-tyrosination and acetylation. While SVC-01 treatment significantly reduced levels of tubulin de-tyrosination, it did not affect levels of tubulin acetylation.

[0305] Figure 34 Adult human cardiomyocytes were treated with vehicle or SVC-01 and collected for analysis of mRNA expression of genes involved in the de-tyrosination-tyrosination cycle. Treatment of adult human cardiomyocytes with SVC-01 did not change expression of genes involved in regulating tubulin de-tyrosination. DETAILED DESCRIPTION

[0306] EXAMPLES

[0307] MATERIALS AND METHODS

[0308] Animals

[0309] Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and performed in accordance with the standards set forth in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

[0310] Rat Cardiomyocyte Isolation and Culture

[0311] Primary adult ventricular cardiomyocytes were isolated from 6- to 8-week-old Sprague Dawley rats or 20- to 30-week-old WKY, ZSFl lean, and obese rats. Briefly, the heart was removed from isoflurane-anesthetized rats and perfused with collagenase solution in a Langendorff apparatus. The digested heart was then minced and triturated using a glass pipette. The resulting supernatant was centrifuged at 300 revolutions per minute (rpm) to isolate the cardiomyocytes, which were then resuspended at low density in rat cardiomyocyte culture medium. The cardiomyocytes were cultured at 37 °C and 5% CO2 with 25 pmol / L cytochalasin D. Rat cardiomyocyte culture medium: Medium 199 (Thermo Fisher 115090) with 1x insulin-transferrin-selenium-X (Gibco 51500056), 1 pg pL Primocin (Invivogen ant-pm-1), 20 mmol / L HEPES (PH 7.4), and 25 pmol / L cytochalasin D. -1 Primocin (Invivogen ant-pm-1), 20 mmol / L HEPES (PH 7.4), and 25 pmol / L cytochalasin D.

[0312] Isolation of Cardiomyocyte Contractility

[0313] Shrinkage force was measured in a custom-built cell chamber (IonOptix) mounted on an LSM Zeiss 880 inverted confocal microscope using a 40 or 63x, 1.4 numerical aperture oil objective and a transmitted light camera (IonOptix MyoCam-S). Experiments were performed at room temperature and field stimulation was provided at 1.0 Hz using a cell stimulator (MyoPacer, IonOptix). After pacing for 10-30 seconds to reach a steady state, five traces were recorded and analyzed. The sarcomere length was optically measured by Fourier transform analysis (IonWizard, IonOptix).

[0314] Western blot

[0315] For whole cell protein extraction, isolated rat cardiomyocytes were lysed in RIPA buffer (Cayman, #10010263) supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling, #5872S) for 1 hour on ice. The supernatant was collected and mixed with 4x loading dye (Li-COR, #928-40004) supplemented with 10% 2-mercaptoethanol and boiled for 8 minutes. The resulting lysate was separated on SDS-PAGE gels and Western blots were performed onto nitrocellulose membranes (Li-COR, #926-31902) using a miniTrans-Blot Cell (Bio-Rad). Membranes were blocked in Odyssey blocking buffer (TBS) (LI-COR, #927-50000) for 1 hour and incubated with the corresponding primary antibody overnight at 4°C. Membranes were washed three times with TBS containing 0.5% Tween 20 (TBST) and incubated with secondary antibody TBS supplemented with additional 0.2% Tween 20 for 1 hour at room temperature. Membranes were washed again with TBST (0.5% Tween 20) and imaged on an Odyssey imager. Image analysis was performed using Image Studio Lite software (LI-COR). All samples were run in duplicate and analyzed in reference to GAPDH.

[0316] Antibody

[0317] Detyrosinated tubulin; rabbit polyclonal antibody (Abeam, ab48389); or affinity purified antibody specifically detecting detyrosinated a-tubulin (Van der Laan, 2019); Western blot: 1 : 1,000.

[0318] a-tubulin; mouse monoclonal, clone DM1A (Cell Signaling, #3873); Western blot: 1 : 1,000.

[0319] GAPDH; mouse monoclonal (VWR GenScript, A01622-40); Western blot: 1 : 1,000.

[0320] Nanoindentation

[0321] Microscale mechanical properties were measured using a nanoindenter (Piuma; Optics 11, Amsterdam, Netherlands) equipped with a dynamic mechanical analysis software package (DMA). Freshly isolated cardiomyocytes were attached to glass bottom dishes coated with NT solution (1 mM Ca 2+ ) of MyoTak (IonOptix). The myocytes were indented to a depth of 1-2 μm using a spherical indenter probe with a radius of 4.5 μm and a stiffness of 0.1 N / m, and then the probe was oscillated in a sinusoidal mode at frequencies of 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, and 50 Hz and an amplitude of 1 μm. Piuma Data Viewer version 2.0 was used to convert the DMA indentation depth and cantilever deflection to E' and E", the magnitude of the elastic modulus was determined by E 2 = E' 2 + E" 2 Assuming a Poisson's ratio (n) of 0.5, and the average value of E was determined as a function of indentation velocity for each experimental condition. Low velocity indentations measure the elastic contribution to stiffness, and high velocity indentations measure both elastic and viscous contributions. The change in modulus with velocity represents the viscoelasticity of the cardiomyocytes, and can be fit to determine the model and degree of viscoelastic behavior.

[0322] Isolated cardiomyocyte stretch and mechanical evaluation

[0323] Freshly isolated or cultured cardiomyocytes were diluted to a sparse density (about 10-fold) in regular Tyrodes solution and placed in a large glass bottom dish (22 x 50 mm) coated with BSA to prevent adhesion of the cardiomyocytes. A small amount of Myotak TMFreshly thawed and place 1.5 pL droplet on a dry area of the culture dish and allow it to polymerize (2-3 min). When the droplet becomes “tacky”, immerse a laser-etched glass rod (Ion Optix LLC) into the solution multiple times to form a thin coating of Myotak, then allow it to dry for 90 seconds before immersing the probe into the cell-containing solution. Then, the cardiomyocytes are attached to the glass struts by gentle tapping and slightly elevated above the coverslip before mechanical testing. The cardiomyocytes are stretched by approximately 10% for either 200 milliseconds (diastolic) or 5 seconds (slow) time intervals using the Myostretcher device (Ion Optix LLC) while recording force and sarcomere length with the IonWizard software and MyoCam imaging system (real-time FFT). Subsequently, each cell is imaged by transmitted light to obtain the cross-sectional area and force is converted to stress. Upon completion of each experimental protocol, the cell struts are lifted from the solution multiple times to remove the cells and the optical fiber is used to clear additional debris. If the cells do not attach well to the struts, the probe is cleaned with Trypsin / EGTA solution, physically cleaned with the optical fiber, and re-coated with Myotak.

[0324] Immunofluorescence

[0325] Cells were fixed in 4% PFA (Electron Microscopy Sciences) for 10 min, washed 3 times with PBS, and permeabilized in 0.1% Triton X-100 for 10 min at room temperature. After washing twice with PBS, cells were placed in blocking buffer (1:1 Seablock (Abeam) and 0.1% Triton X-100 (Bio-Rad) in PBS) for at least 1 h at room temperature, then labeled with primary antibodies (see below) for 24-48 h at 4°C. Cells were then washed three times with TBS, then labeled with secondary antibodies (see below) for 2-4 h at room temperature in TBS, and finally washed twice with TBS. Stained cells were mounted on a #1.5 coverslip in Prolong Diamond Antifade Mountant (Thermo Fisher) for imaging. Alternatively, cells were imaged directly in a chamber with TBS. Imaging was performed on a Zeiss 880 Airyscan confocal microscope running on an Axiovert Z1 inverted microscope equipped with a Plan-Apochromat 63x oil objective (numerical aperture 1.4). Image analysis used ZEN Black software for Airyscan processing, which involves signal integration from 32 individual sub-resolution detectors in the Airyscan detector, and subsequent deconvolution processing of this integrated signal.

[0326] transthoracic echocardiography

[0327] Echocardiographic measurements were acquired using a Vevo 2100 ultrasound system (Visual Sonics Inc., Toronto, Ontario, Canada) with MS250 (13-24 MHz) sensor. Animals were sedated with inhaled isoflurane (2-3%). Parasternal long axis, parasternal short axis, apical four chamber, and apical two chamber views were acquired to assess cardiac structure and function. Diastolic function was assessed using pulsed wave and tissue Doppler imaging. The protocol used was based on recommendations of the European Society of Cardiology Working Group on Myocardial Function (Zacchigna S et al., 2021).

[0328] Invasive hemodynamics

[0329] Animals were induced with isoflurane (5%) prior to cannulation and mechanical support. Anesthesia plane was maintained using isoflurane (2-3%). A 2-French pressure-conductance catheter (Transonic Systems Inc., Ithaca, New York, USA) was calibrated using left ventricular volumes measured by ultrasound. The right common carotid artery was then cut open and the catheter was advanced retrograde into the left ventricle as previously described. Data were acquired using PowerLab and LabChart Pro (ADInstruments) and analyzed offline using LabChart Pro (ADInstruments).

[0330] Decayylated tubulin immunofluorescence

[0331] Formalin-fixed paraffin-embedded (FFPE) sections on slides were deparaffinized by washing with xylene and decreasing concentrations of ethanol (100%, 95%, 75%, and 50%) and ddH2O, sequentially. Sections were placed in 1x Reveal Retrieval Solution (Biocare Medical, Cat# RV1000M) for antigen retrieval. Antigen retrieval was performed using a pressure cooker (Instant Pot Pro 10-in-1 Pressure Cooker, 8 quart, Amazon.com) under high pressure for 15 minutes. After cooling, sections were subjected to a second round of antigen retrieval under high pressure in a pressure cooker in 1x Antigen Unmasking Solution (Citrate-based) (Vector Laboratories, Cat# H-3300-250) for 15 minutes. Sections were then washed with ddH2O, followed by PBS. Sections were permeabilized with PBS + 0.25% Triton X-100 and then incubated in fish serum blocking buffer containing 0.1% Tween 20 (Thermo Scientific, Cat# 37527) for 1 hour at room temperature.

[0332] Slices were co-stained with mouse monoclonal (B-5-1-2) anti-a-tubulin antibody (Sigma-Aldrich, Cat# T5168; 1 :50) and rabbit polyclonal de-tyrosinated a-tubulin antibody (ab48389, Lot GR3425171; 1 :50) in blocking buffer for 2 days at room temperature. Slices were washed in PBS containing 0.1% Tween 20 (PBST) and then incubated with goat anti-rabbit antibody (coupled to Alexa Fluor 647) and with goat anti-mouse secondary antibody (coupled to Alexa Fluor 568) and WGA coupled to Alexa Fluor 488 (Invitrogen, Cat# W11261; final concentration 25 pg / mL) in PBST for 2 days at room temperature. Slices were then stained with Hoechst 33342, trihydrochloride, trihydrate (Invitrogen, Cat# H3570, final concentration 10 pg / mL in PBS) and mounted with Prolong Diamond Antifade Mountant (Invitrogen, Cat# P36961).

[0333] Longitudinally aligned cardiomyocytes were identified within the myocardium. Myocardial regions were imaged on a Zeiss Airyscan 880 microscope using a 63x oil immersion objective (numerical aperture of 1.4) with four-channel Z-stack scanning (4x500nm slices) with a pixel size of 18.3nm x 18.3nm. Images were background subtracted in FIJI (NIH) and maximum intensity projections were generated. Using the WGA and Hoechst channels, rectangular ROIs were blindly drawn within the cytoplasmic portion of individual cardiomyocytes, while excluding the nucleus and perinuclear regions. From these ROIs, a-tubulin and de-tyrosinated microtubule images were extracted. Using the FIJI plugin trainable WekaSegmentation, a set of preliminary a-tubulin and de-tyrosinated microtubule images were used to train a classifier model for each channel. The final classifier model was applied to all images in the dataset for each channel to generate binary maps of microtubule networks. Microtubule density was determined by calculating the area coverage percentage of the binary map within each ROI.

[0334] Cathepsin B assay

[0335] Screening kit from Merck (Ref: MAK200) according to the manufacturer’s recommendations.

[0336] Nanostring mRNA analysis

[0337] Two custom NanoString code sets; one for tubulin-associated transcripts (tubulin isoforms, modifying enzymes, and microtubule-associated proteins) and the other for heart failure- and extracellular matrix-associated transcripts, along with three housekeeping genes (Gapdh, Rpl4, and Tbp), were designed with assistance from NanoString Technologies as previously described. Nineteen isolated mRNAs were evaluated for concentration (>20 ng / μL) and quality (OD 260 / 230 > 1.8) using a BioTek Synergy 4 microplate reader. A total of 200-300 ng of RNA was sent to the Wistar Institute Core for processing, and the levels of target tubulin-associated mRNAs were determined by NanoString Technologies’ nCounter Custom CNV detection method. Subsequently, RNA quantities were determined on the nCounter Digital Analyzer according to the manufacturer’s protocol, and the barcode counts were tabulated into a file for analysis in the nSolver Analysis software (NanoString 4.0). The internal negative controls contained in the NanoString Prep were used to subtract background, and transcript abundances below background levels were excluded from analysis. Data were then normalized to the geometric mean of the housekeeping genes (Gapdh, Rpl4, and Tbp) using the nSolver software package, and exported for statistical analysis. Significance was assessed by unadjusted paired t-tests using OriginPro 2019.

[0338] Human myocardial tissue procurement

[0339] Human hearts from failing hearts were procured at the time of orthotopic heart transplantation at the University of Pennsylvania Hospital after prospective informed consent of all participants. Non-failing hearts were obtained from cadaveric donors at the time of organ donation. Consent for use of donor heart tissue for research was obtained from next of kin. In all cases, the heart was arrested in situ using ice-cold cardioplegia solution and transported to the laboratory on wet ice.

[0340] Human myocardial cell isolation

[0341] Once excised, the heart is transported from the operating room to the laboratory in cold cardioplegic solution. Myocyte isolation is performed as previously described. The apex wedge is cut from the free wall of the left ventricle, and a catheter is placed into the left anterior descending branch. The cannula is flushed with cold saline to assess perfusion of the wedge and to ligate any major blood vessels. The myocardium is then perfused with warm non-recirculated Krebs-Henseleit Buffer (KHB) solution (12.5 mM glucose, 5.4 mM KC1, 1 mM lactate, 1.2 mM MgS04, 130 mM NaCl, 1.2 mM NaH2P04, 25 mM NaHC03, 2 mM sodium pyruvate, 20 mM BDM, and 10 mM taurine, pH 7.4) for 10 minutes. Then, the myocardium is perfused with recirculating KHB and collagenase type II (294 units / mL) (Worthington Biochemical Corp., Lakewood, NJ, USA) for 5 minutes, followed by the slow reintroduction of calcium to achieve a final concentration of 1 mM. Depending on the manner of myocardial digestion, the collagenase and calcium-supplemented KHB is recirculated for 25-30 minutes. The tissue is then removed from the cannula, rinsed in KHB containing 1% bovine serum albumin (BSA) to stop the digestion, and then minced. The cell suspension is then filtered using a 280-uM nylon mesh, centrifuged (25 x g, 2 minutes), and resuspended in regular Tyrode's solution. The cells are treated with VASH inhibitor (VASHi) (1 mM) or vehicle (DMSO) and then placed in an incubator for 4 hours.

[0342] Human muscle cell contractility

[0343] Contractility is assessed as previously described. After 4 hours of incubation with VASHi or vehicle, the cardiomyocytes are transferred to 35 mm glass-bottomed dishes. Contractility data are collected at 37°C. The myopacer (IonOptix MYP100) is used to stimulate the cardiomyocytes at 0.5 Hz with a custom-made carbon electrode inserted into the glass-bottomed dish. The cells are stimulated for 10-15 seconds to reach steady-state contraction, and then data are recorded using high-speed video imaging with a Nikon PU-2000 inverted microscope (IonWizard, IonOptix) with a 40x objective. At least 5 contractions are recorded for each myocyte, and analyzed offline.

[0344] Results and discussion

[0345] Effective targets for heart failure therapy

[0346] Proteomic analysis of myocardial tissue collected from a cohort of HCM patients who underwent septal myectomy for symptomatic LVOTO showed that many abnormalities in HCM patients were similar in patients with and without identified sarcomeric protein mutations (Schuldt M et al., Circ Heart Fail. 2021; 14: 39-55). However, notable exceptions were abnormalities in the abundance of cytoskeletal proteins, particularly de-tyrosinated a-tubulin, which were much higher in patients with HCM and known sarcomeric gene mutations than in patients without identified mutations. This particular finding is notable because recent studies have shown that this particular post-translational modification, de-tyrosination of a-tubulin, has a significant impact on the stability and density of the cardiomyocyte cytoskeleton and on cell biomechanics. This particular increase in de-tyrosination and associated changes in the microtubular network of cardiomyocytes are causally related to increased stiffness and viscoelasticity, which reduces contractility and slows both systole and diastole. As part of adaptive and pathological cardiac remodeling, microtubules are extensively altered, which has different effects on the structure and function of cardiomyocytes.

[0347] Distribution of tubulin de-tyrosination in myocardial tissue

[0348] Therefore, we sought to describe the distribution of tubulin de-tyrosination in myocardial tissue from healthy animals Figure 1 ). Using our affinity-purified antibody that specifically detects de-tyrosinated a-tubulin (Van der Laan et al., 2019), we optimized the immunohistochemical staining of formaldehyde-embedded left ventricular heart tissue. While microtubular densification has been shown (Cheng G. et al., 2008) to be responsible for increased hypertrophy and stiffness, healthy ventricular cardiomyocytes showed a typical homogenous staining pattern with longitudinal and transversal de-tyrosinated microtubules, as previously shown on isolated cardiomyocytes (Robison et al., 2016). Notably, we could also see an increased level of staining in the outer layers of the heart tissue.

[0349] Design and synthesis of specific VASH compounds (SVCs)

[0350] Since no small molecule has been designed so far to inhibit VASH, the enzyme responsible for tubulin de-tyrosination, we designed various compounds based on the natural substrate of this enzyme. We performed a broad analysis of the human proteome and sought to identify human proteins that encode a-EEY sequences at the C-terminus. Only 3 proteins fit the criteria, including a-tubulin. Therefore, we developed 5 compounds that all structurally have a tyrosine, which is crucial for specificity Figure 2 ). In the context of an in vitro de-tyrosination assay, we used recombinant protein human VASH1 Figure 3) and their natural substrates. Using iterative cycles of computer-aided drug discovery and medicinal chemistry scrutiny followed by ELISA (in vitro) and HCS (intracellular) testing, we have collected a SAR database containing new chemical entities. The potency and selectivity of these compounds are considered to meet the criteria of further drug development. Our initial prototype inhibitor (Epo-Y) showed dose-dependent inhibition of des-tyrosination enzyme activity. That is, a newly developed specific VASH compound (SVC) named SVC_01 showed about 100-fold inhibition compared to Epo-Y. The prodrug SVC_02 is inactive against the therapeutic target and presentation of SVC_02 to the reaction mixture does not result in decreased activity. Next, using a human cell line, we assayed the activity of compound SVC_01 in a cellular system, which further indicates penetration and cellular potency. After 2 hours of human cell exposure to the SVC_01 compound, we fixed and analyzed the labeling of des-tyrosinated tubulin, as well as another post-translational modification called acetylation Figure 4 ) by specific immunofluorescence. We compared the staining levels to a human VASH double-knockout cell line, in which the levels of des-tyrosinated microtubules were undetectable. Our specific VASH compound, SVC_01, completely reduced the levels of tubulin des-tyrosination in human cells without affecting acetylation levels, further demonstrating the specificity of the approach. Despite the specificity, micromolar concentrations of SVC_01 were used to obtain complete inhibition. To improve the cellular IC 50 of SVCs, we designed compounds with better cellular penetration properties. In turn, we obtained SVC_02 and SVC_03, which both act as prodrugs and are ineffective against the recombinant VASH1 enzyme but highly potent (low nanomolar IC 50 )( Figure 5 ) in human cells. While the substrate is fairly unique in the human proteome (ending in -EEY), we performed a specificity analysis by selecting different cysteine proteases and testing their activity in the presence of SVCs. We selected proteases according to different proteolytic mechanisms.

[0351] The following are details of the selection criteria:

[0352] - Proteases with documented carboxypeptidase activity against a-tubulin. Carboxypeptidase A hydrolyzes the peptide bond of C-terminal residues with aromatic or aliphatic side chains Figure 6 .

[0353] - Cysteine proteases, preferably with a non-aromatic residue at P1’. The optimal recognition site of the human rhinovirus (HRV) type 14 3C protease is LEVLFQ / GP, with a glycine at the P1’ positionFigure 7 ).

[0354] - Cysteine proteases, preferably P1’ site is aromatic residue. Similar to VASH, Cathepsin B prefers an aromatic residue at the P1’ site. This is considered the best control for specificity Figure 8 ).

[0355] None of these proteases were inhibited in the presence of SVC in the high micromolar range, further supporting the specificity of these compounds for the therapeutic target VASH1.

[0356] SVC has a superior safety profile compared to microtubule binding drugs

[0357] Next, we performed safety pharmacology experiments and head-to-head compared the cytotoxicity of SVC on human cells. For comparison, we used a microtubule binding drug, parthenolide, which was previously thought to be a de-tyrosination inhibitor (Fonrose et al., 2007). The compound was later shown to disrupt microtubules by non-specific binding to tubulin in the molecular structure (Hotta T. et al., 2021). Using human cell lines, we observed severe cell survival issues in the micromolar concentration range when using parthenolide and paclitaxel Figure 9 ). On the other hand, no toxicity was observed for SVC_01 even after several days of exposure at concentrations as high as 100 micromolar. Mitochondrial health was determined using Seahorse equipment XF. Seahorse XF Analyzer measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in multi-well plates, detecting key cellular functions such as mitochondrial respiration and glycolysis. The XF Analyzer can perform compound addition and mixing, label-free analysis detection, and automatic measurement of OCR and ECAR in real time. No mitochondrial defects were measured at 20 micromolar concentration of our VASH inhibitor Figure 10 ). Considering the application of our active pharmaceutical ingredient (API) in the heart, we also performed a hERG analysis with our SVC. The human ether-a-go-go-related gene (hERG) encodes the cardiac inward-rectifier voltage-gated potassium channel (IKr) involved in cardiac repolarization. No potential cardiotoxicity was identified in the dose response in the presence of SVC_03 Figure 12 ).

[0358] Concept and proof of efficacy in preclinical models

[0359] To perform a proof of efficacy, we provided SVC_01 to primary cardiomyocytes isolated from Sprague Dawley rats. As we previously found in human cells Figure 4 and Figure 5), a dose-dependent inhibition of tubulin de-tyrosination was observed, with a decrease close to 80% ( Figure 12 ) after 2 hours of incubation with SVC_01. This finding further confirms that VASH is the main de-tyrosinase in these cell types and that our newly designed specific VASH compound is very efficient in binding the target. Next, we performed functional studies by measuring key cardiomyocyte parameters, including resting, fractional shortening, contraction time and relaxation time. We observed a dose-dependent and statistically significant improvement in contraction and relaxation times in the presence of SVC_01 ( Figure 13 ). The ZSF1 rat model is a cross-bred rat obtained by crossing ZDF females and SHHF males. This model is an established HFpEF rat model. Obese ZSF1 (O-ZSF1) cross-bred rats are compound heterozygotes with two different mutant alleles encoding for a defective leptin receptor, leading to loss of receptor function. As a consequence, these animals have increased food intake and develop obesity, diabetes and the prominent features of HFpEF resulting thereof. ZSF1 rats inheriting two wild-type alleles or only one parental mutant allele have normal leptin signaling, balanced food intake and energy expenditure, normal body weight and do not develop disease features (L-ZSF1). We performed Western blot analysis on cardiomyocyte tissue prepared from Wistar Kyoto (WKY), ZSF1 lean and ZSF1 obese rats ( Figure 14 ). In line with previously published data on microtubule densification and tubulin de-tyrosination, we found an increase in both total tubulin and de-tyrosinated tubulin in ZSF1 obese rat cardiomyocyte tissue compared to lean and WKY control groups. Isolated cardiomyocytes from ZSF1 rats incubated with SVC_01 showed a significant reduction in tubulin de-tyrosination ( Figure 15 ). Moreover, treatment with our VASH inhibitor improved functional parameters of isolated diseased cardiomyocytes of 30-week-old obese ZSF1 rats. Both contraction and relaxation kinetics were improved after 2 hours of treatment with the compound. The beneficial effect of treatment was similar to that obtained with cardiomyocytes overexpressing TTL (the reverse enzyme responsible for re-tyrosination, thus reducing de-tyrosination) by adenoviral transfection ( Figure 16 ). Functional improvements were statistically significant and importantly, cardiomyocytes isolated from obese rats treated with the vehicle showed a clear functional impairment. Similar results were observed by assessing the lateral stiffness. In fact, both VASHi treatment as TTL overexpression led to a significant and strong functional improvement in both lateral and longitudinal stiffness of cardiomyocytes ( Figure 17 , Figure 18). To test the efficacy of treatment in reducing clinically relevant cardiac events, we injected compounds (dose 6.6 mg / kg) in the tail vein of Sprague dawley rats and collected heart tissue for Western blot analysis. Eight hours after intravenous injection, a clear and significant reduction of tubulin de-tyrosination was observed in myocardial tissue ( Figure 19 ). The same route of administration was used for the ZSF1 rat model, allowing the therapeutic target to bind to heart tissue. We found a ~60% reduction of tubulin de-tyrosination in lean and obese animals 8 hours after intravenous injection of SVC_02 ( Figure 20 ). The main results were a significant reduction of Tau, mitral deceleration time and a trend for a significant reduction of LVDEP after acute intravenous injection of VASHi ( Figure 21 ).

[0360] Peak exercise tolerance was poorer and the rate-dependent increase in filling pressures was steeper in response to stress stimuli in ZSF1 obese rats. SVC reduced microtubule de-tyrosination in the heart of ZSF1 obese rats. This resulted in faster relaxation as judged by a significant improvement in mitral deceleration time (A) and left ventricular relaxation time constant TAU (B), a trend for a reduction of spontaneous LVEDP and a mild inducible increase in LVEDP. Thus, overall, these results validate the effectiveness of ZSF1 rats as a model of HFpEF and importantly demonstrate that reducing microtubule de-tyrosination using SVC has therapeutic potential in mitigating diastolic impairment in HFpEF and is an effective strategy to prevent or treat heart failure.

[0361] To determine whether changes in extracellular matrix (ECM) or microtubule network related transcripts were consistent with increased myocardial stiffening and microtubule network stability, we performed Nanostring analysis with a custom gene expression panel. Among extracellular matrix proteins, Vcan, Fn1 and Collal were significantly increased in isolated cardiomyocytes of obese ZSF1 rats compared to lean ZSF1 rats ( Figure 22 ). Among microtubule network proteins and regulators, Tubb2a, Vash1, Hdac6 and Map1a were significantly increased in obese ZSF1 rats compared to lean ZSF1 rats.

[0362] Intravenous delivery of SVC-02 significantly reduced de-tyrosinated tubulin abundance in ZSF1 rat myocardium ( Figure 20 ). This reduction could also be observed by super-resolution immunofluorescence imaging of fixed LV tissue sections from obese animals treated with vehicle and SVC-02 as VASHi ( Figure 23). As expected, tyrosinated microtubule density was significantly reduced in obese animals treated with VASHi (SVC-02) compared to vehicle treatment Figure 24 ). Other studies confirmed that obese rats have three-fold higher levels of tyrosinated tubulin compared to WKY and lean controls Figure 25 ), and levels of tyrosination were reduced to lower levels after treatment with SVC-02.

[0363] Human cardiomyocytes were isolated from failing (N=4) and non-failing (N=3) human hearts and then treated with vehicle or VASHi (SVC-02) to assess the biological activity of the small molecule compound in primary human cardiomyocytes. Patient demographics are detailed in the table Figure 26 ). The summary traces for all non-failing and failing patients are shown in the figure Figure 27 ). SVC-02 shortened the time to 50% relaxation, which is consistent with the observations in isolated cardiomyocytes and in vivo in obese rats Figure 28 . Notably, SVC-02 did not significantly change the fractional shortening, the velocity of contraction, or the resting sarcomere length of failing human cardiomyocytes.

[0364] To further determine the safety profile of the compound in an animal model, we administered SVC-02 to Swiss mice and analyzed different organ structures including kidney, heart, and lung by immunohistological description. Treatment with the specific VASH inhibitor (SVC-02) did not alter organ structures Figure 29 ), nor did it cause obvious tissue damage.

[0365] Further characterization of the specific VASH compound also showed that using a high content screening method in cells, the IC 50 of SVC-06 was very low (2.1 nM) Figure 30 . In addition, oral administration of SVC-06 to Sprague-Dawley rats resulted in a significant reduction of tubulin tyrosination levels in myocardial tissue. Quantification of the western blot signals showed that tubulin tyrosination levels were reduced by more than 40% after a single oral administration of SVC-06 to Sprague-Dawley rats Figure 31 .

[0366] SVC-01 was assayed for specificity using human VASH1 and VASH2 recombinant enzymes. We found that SVC-01 effectively inhibited the tyrosination activity of both enzymes Figure 32 . In addition, when adult human cardiomyocytes were treated with SVC-01, we found that tubulin tyrosination was significantly reduced, while other post-translational modifications such as acetylation remained unchanged Figure 33). These results demonstrate the specificity of the target and mechanism of action of the developed compounds. Finally, to ensure that the regulation of other genes involved in the detyrosination-tyrosination cycle was not affected, we analyzed the gene expression of all relevant and associated genes in adult cardiomyocytes after SVC-02 treatment. We found no change in the expression of the VASH1, VASH2, MATCAP, TTL, or SVBP genes between the vehicle control and SVC-02 treated cells Figure 34

[0367] References

[0368] Aillaud, C. et al. Vasohibins / SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science 358, 1448-1453 (2017).

[0369] Caporizzo, M. A., Chen, C. Y. & Prosser, B. L. Cardiac microtubules in health and heart disease. Experimental Biology and Medicine 260, 1535370219868960 (2019).

[0370] Chen, C. Y. et al. Suppression of detyrosinated microtubules improves cardiomyocyte function in human heart failure. Nature Medicine, volume 24, pages 1225-1233 (2018), doi.org / 10.1038 / s41591-018-0046-2.

[0371] Chen, C. Y. et al. Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes. Circulation Research 116, 2662 (2020).

[0372] ​Cheng G. et al., A direct test of the hypothesis that increased microtubule network density contributes to contractile dysfunction of the hypertrophied heart. Am J Physiol Heart Circ Physiol. 2008 May; 294(5): H2231-41. doi: 10.1152 / ajpheart.91515.2007.

[0373] Fonrose et al., (2007). “Parthenolide inhibits Tubulin Carboxypeptidase Activity”, Cancer research, vol. 67, n° 7, 1 April 2007, pages 3371-3378.

[0374] Hotta T. et al., Parthenolide Destabilizes Microtubules by Covalently Modifying Tubulin. Curr Biol. 2021 Feb 22; 31(4): 900-907.e6. doi: 10.1016 / j.cub.2020.11.055.

[0375] Kerr, J. P. et al. Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle. Nat Commun 1-14 (2015). doi: 10.1038 / ncomms9526

[0376] McDonagh T. A et al., 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2021 Sep 21; 42(36): 3599-3726. doi: 10.1093 / eurheartj / ehab368.

[0377] Maron, B.J. et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation. 1995 Aug 15;92(4):785-9. doi: 10.1161 / 01.cir.92.4.785.

[0378] Robison, P. et al. Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes. Science 352, aaf0659-aaf0659 (2016).

[0379] Roll-Mecak A., The Tubulin Code in Microtubule Dynamics and Information Encoding, Dev Cell, 2020 Jul 6; 54(1): 7-20. doi: 10.1016 / j.devcel.2020.06.008.

[0380] Schuldt, M. & Kuster, D. W. D. Proteomic and functional studies reveal detyrosinated tubulin as treatment target in sarcomere mutation-induced hypertrophic cardiomyopathy. Circulation: Heart Failure (2020).

[0381] Tsutsui H. et al., Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science, 1993 Apr 30; 260(5108): 682-7. doi: 10.1126 / science.8097594.

[0382] Van der Laan S. et al., Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination. Cell Reports. Volume 29, Issue 12, 17 December 2019, Pages 4159-4171.e6. doi.org / 10.1016 / j.celrep.2019.11.074.

[0383] Yu X. et al., MARK4 controls ischaemic heart failure through microtubule detyrosination. Nature, volume 594, pages 560-565 (2021). doi:10.1038 / s41586-021-03573-5

[0384] Zacchigna S, Paldino A, Falcao-Pires I, Daskalopoulos EP, Dal Ferro M, Vodret S, Lesizza P, Cannata A, Miranda-Silva D, Lourenco AP, et al. Towards standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on Myocardial Function. Cardiovasc Res. 2021; 117: 43-59. doi:10.1093 / cvr / cvaa110.

Claims

1. A compound of formula (I): Or its pharmaceutically acceptable salts and / or solvates, in X is or -NH-CH2- R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl, R is O-R 2 wherein R 2 is a C1-C6 aliphatic chain, which aliphatic chain is optionally substituted, or a C1-C6 alkyl-aryl, R 3 is OH or O-Ci-C6aliphatic chain, Y is -(CH2) m - wherein m = 2 or R 5 is C(O)OH or O-Ci-C6-alkyl, and R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain, Its use for the prevention and / or treatment of heart failure, cardiomyopathy, cardiac dysfunction induced by myocardial infarction and / or for improving cardiac function in subjects in need.

2. The compound of formula (I) according to claim 1, wherein the compound of formula (I) is formulated into a pharmaceutical composition with a pharmaceutically acceptable excipient.

3. The compound of formula (I) according to claim 2, wherein the pharmaceutical composition is for oral or parenteral administration.

4. The compound of formula (I) according to any one of claims 1 to 3, wherein in the compound of formula (I), X is R 1 is or NR 1a R 1b wherein R 1a is H and R 1b is C1-C6 alkyl unsubstituted or substituted by C(O)-O-C1-C6 alkyl or C(O)-NH-C1-C6 alkyl, R is O-R 2 wherein R 2 is a C1-C6aliphatic chain, which aliphatic chain is unsubstituted or substituted by phenyl, or C1-C6alkyl-aryl, R 3 is OH or O-Ci-C6aliphatic chain, Y is -(CH2) m - wherein m = 2 or R 5 is C(O)OH or O-Ci-C6-alkyl, and R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain.

5. The compound of formula (I) according to any one of claims 1 to 3, wherein in the compound of formula (I), X = -NH-CH2- R 1 is NR 1a R 1b wherein R 1a is H and R 1b is C1-C6alkyl substituted with C(O)-NH-C1-C6alkyl, R is O-R 2 wherein R 2 is Ci-C6alkyl-aryl, and R 3 is O-C1-C6aliphatic chain.

6. The compound of formula (I) according to claim 4, wherein in the compound of formula (I), R 1 is and R is O-R 2 wherein R 2 is a C1-C6 aliphatic chain, which is unsubstituted or substituted by phenyl, R 3 is OH or O-Ci-C6aliphatic chain, Y is -(CH2) m - wherein m = 2 or R 5 is C(O)OH or O-Ci-C6-alkyl, and R 6 is NH-CH(R 7 )-(CH2) n -R 8 wherein R 7 is H, and R 8 is aryl, and n is 1, 2 or 3, preferably 1, or O-C1-C6 aliphatic chain.

7. The compound of formula (I) according to any one of claims 1 to 6, wherein the compound of formula (I) is selected from the group consisting of: Its pharmaceutically acceptable salts and / or solvates, and Its associated objects.

8. The compound of formula (I) according to any one of claims 1 to 7, wherein the compound of formula (I) improves the dynamics of failing cardiomyocytes, including increasing diastolic velocity, increasing contractile force, and / or reducing viscoelasticity.

9. The compound of formula (I) according to any one of claims 1 to 8, wherein the subject in need suffers from heart failure with reduced ejection fraction (HFrEF).

10. The compound of formula (I) according to any one of claims 1 to 8, wherein the subject in need suffers from heart failure with preserved ejection fraction (HFpEF).

11. The compound of formula (I) according to any one of claims 1 to 10, wherein the compound of formula (I) is administered in combination with current standard treatment for cardiac dysfunction induced by heart failure, cardiomyopathy or myocardial infarction to a subject in need.

12. A combination product comprising: a) the compound of formula (I) according to any one of claims 1, 4 to 7, or the pharmaceutical composition according to claim 2 or 3, and b) Current standard of care for cardiac dysfunction induced by heart failure, cardiomyopathy, or myocardial infarction.

13. Use of the compound (I) or a conjugate thereof according to any one of claims 1 to 7, said conjugate comprising a fragment of compound (I) linked or not linked to an optionally labeled biomolecule, as a research tool for research and development activities on cardiac dysfunction or heart failure, particularly selected from: - In vitro methods are used to study the role of microtubule detyrosineing in heart diseases, such as their occurrence, invasiveness, and progression. - and related kits for performing the screening assays and methods.

14. Use of a conjugate comprising a fragment of a compound of formula (I) as defined in any one of claims 1 to 7, said fragment being linked to a biomolecule selected from a polypeptide, a protein, a biomarker, a photo-labeling agent such as rhodamine, a coumarin derivative, a cyanine derivative or fluorescein, an affinity probe such as biotin, or an E3 ubiquitin ligase recruiting agent such as thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096 or d9A-2.

15. Use of a conjugate comprising a fragment of a compound of formula (I) as defined in any one of claims 1 to 7, said fragment being linked to a biomolecule selected from a polypeptide, a protein, a biomarker, a photo-labeling agent such as rhodamine, a coumarin derivative, a cyanine derivative or fluorescein, an affinity probe such as biotin, or an E3 ubiquitin ligase recruiting agent such as thalidomide, VH032, VH101, dBET1, dFKBP12, QCA570, ZNL-02-096 or d9A-2, for the preparation of a medicament for the treatment of a disease.

16. Use of a conjugate comprising a fragment of a compound of formula (I) as defined in any one of claims 1 to 7

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